Multicomponent Nucleic Acid Enzymes (MNAzymes) are made of enzymatically inactive subunits that assemble to form an active enzyme in the presence of an assembly facilitator (see US 2011/0143338; US 2007/0231810; WO/2008/122084; WO/2007/041774; and Mokany et al., J Am Chem. Soc. 2010 Jan. 27; 132(3): 1051-1059, each of which is incorporated by reference in its entirety). Because the inactive subunits form the active enzyme in the presence of the assembly facilitator, but remain separate and inactive in its absence, enzyme activity is specifically activated by the presence of the assembly facilitator. The active enzyme can generate a detectable signal that indicates the presence of the assembly facilitator, or catalyze another desired reaction. In general, the inactive subunits, or partzymes, each include a sensor arm, which interacts with the assembly facilitator, and may also include a substrate recognition arm which interacts with the enzyme's substrate.
In one particularly useful configuration, the assembly facilitator is a nucleic acid, and the partzymes each include a sensor arm containing part of a complementary nucleic acid, such that base pairing brings the inactive subunits together to form the active enzyme. The active enzyme may cleave a substrate that contains a fluorophore and a matched quencher, resulting in increased fluorescence and permitting qualitative or quantitative detection of the assembly facilitator. For example, the MNAzyme may be utilized for quantitative detection of a nucleic acid produced during a real-time PCR reaction. The substrate may also be a nucleic acid, and the active enzyme may include substrate recognition arms containing complementary nucleic acids which facilitate cleavage of the substrate.
In addition to recognizing the assembly facilitator and substrate through nucleic acids, the active enzyme may itself be made up of catalytically active nucleic acid molecules (and thus, some MNAzymes may be made entirely of nucleic acids). As is well known in the art, nucleic acid molecules can adopt secondary structural configurations which can confer enzymatic or catalytic activity. In vitro evolution technology has facilitated the discovery and development of such catalytic nucleic acids, often referred to as “DNAzymes” or “ribozymes,” that are capable of catalyzing a broad range of reactions including cleavage of nucleic acids (Carmi et al, 1996; Raillard and Joyce, 1996; Breaker, 1997; Santoro and Joyce, 1998), ligation of nucleic acids (Cuenoud and Szostak, 1995), porphyrin metallation (Li and Sen, 1996), and the formation of carbon-carbon bonds (Tarasow et al, 1997), ester bonds (Illangasekare et al, 1995) or amide bonds (Lohse and Szostak, 1996).
In particular, DNAzymes and ribozymes have been characterized which specifically cleave distinct nucleic acid sequences after hybridizing via Watson Crick base pairing. DNAzymes are capable of cleaving either RNA (Breaker and Joyce, 1994; Santoro and Joyce, 1997) or DNA (Carmi et al, 1996) molecules. Catalytic RNA molecules (ribozymes) are also able to cleave both RNA (Haseloff and Gerlach, 1988) and DNA (Raillard and Joyce, 1996) sequences. The rate of catalytic cleavage of most nucleic acid enzymes is dependent on the presence and concentration of divalent metal ions such as Ba2+, Sr2+, Ca2+, Ni2+, Co2+, Mn2+, Zn2+, and Pb2+ (Santoro and Joyce, 1998; Brown et al, 2003).
Catalytic nucleic acids, such as the hammerhead ribozyme and the 10:23 and 8:17 DNAzymes, have multiple domains. They have a conserved catalytic domain (catalytic core) flanked by two non-conserved substrate binding domains (“hybridizing arms”), which are regions of sequence that specifically bind to the substrate. Haseloff and Gerlach engineered the hammerhead ribozyme, which was so named for the stem-loop structure that brings the two conserved domains together forming the catalytic core (Haseloff and Gerlach, 1988). The “10:23” and “8:17” DNAzymes are capable of cleaving nucleic acid substrates at specific RNA phosphodiester bonds (Santoro and Joyce, 1997). The 10:23 DNAzyme has a catalytic domain of 15 deoxynucleotides flanked by two substrate-recognition arms. The 8:17 DNAzyme has a catalytic domain of 14 deoxynucleotides that is also flanked by two substrate-recognition arms.
A catalytic nucleic acid can cleave a nucleic acid substrate with a target sequence that meets minimum requirements. The substrate sequence should be substantially complementary to the hybridizing arms of the catalytic nucleic acid, and the substrate should contain a specific sequence at the site of cleavage. Specific sequence requirements at the cleavage site include, for example, a purine:pyrimidine ribonucleotide sequence for cleavage by the 10:23 DNAzyme (Santoro and Joyce, 1997), and the sequence uridine:X for the hammerhead ribozymes (Perriman et al., 1992), wherein X can equal A, C, or U, but not G.
Catalytic nucleic acids have been shown to tolerate only certain modifications in the area that forms the catalytic core (Perreault et al., 1990; Perreault et al., 1991; Zaborowska et al., 2002; Cruz et al., 2004; Silverman, 2004)). Examples of sequences responsible for catalytic activity of DNAzymes are listed in Table 1.
TABLE 1Exemplary sequences for some activeDNAzymes and their substratesDNAzymetypeDNAzyme sequenceSubstrate sequence 8:17(N)xTNNNAGCNNNWCGK(N)x(N′)x (rN)x G (N′)x(SEQ ID NO: 152 and SEQ ID NO: 154)10:23(N)xGGMTMGHNDNNNMGD(N)x(N′)x rR rY (N′)x(SEQ ID NO: 153)N = A, C, T, G or any analogue; N′ = any nucleotide complementary to N; (N)x or (N′)x = any number of nucleotides; W = A or T; K = A, G or AA; rN = any ribonucleotide base; (rN)x = any number of ribonucleotides; rR = A or G; rY = C or U; M = A or C; H = A, C or T; D = G, A or T
The substitution of certain deoxyribonucleotides for certain ribonucleotides in known ribozymes has been attempted under certain conditions (McCall et al., 1992). Ribozymes that have been fully converted into DNA have had no activity, apparently due to the conformational differences of RNA and DNA (Perreault et al., 1990). These studies demonstrate that RNA enzymes cannot be modified into working DNA enzymes by merely replacing ribonucleotides with deoxyribonucleotides.
There have been some studies which attempted to develop certain homodimeric or heterodimeric ribozymes for therapeutic applications (Kuwabara et al., 1999; Kuwabara et al., 2000; Oshima et al., 2003). In those studies, the catalytic core of the ribozyme comprised solely of ribonucleotides. Moreover, the capacity for DNAzymes to function in dimeric or multimeric formats has not been considered, nor has any information been provided as to how to extrapolate from a dimeric ribozyme to a dimeric DNAzyme in terms of a possible structure of a dimeric DNAzyme and resulting activity.
Catalytic nucleic acids have been used in combination with in vitro amplification protocols as a means of generating a detectable signal, thus allowing real time monitoring of amplified nucleic acid target sequences (Todd et al., 2000) (U.S. Pat. No. 6,140,055; U.S. Pat. No. 6,201,113; WO 99/45146; PCT/IB99/00848; WO 99/50452). Zymogene detection (U.S. Pat. No. 6,140,055; U.S. Pat. No. 6,201,113; WO 99/45146; PCT/IB99/00848; WO 99/50452), also known in the art as DzyNA detection (Todd et al., 2000), results in concurrent target and signal amplification. This occurs because the catalytic DNAzymes or ribozymes co-amplify along with target sequences to produce amplicons that function as true enzymes capable of multiple turnovers. As such, each catalytic nucleic acid amplicon cleaves multiple reporter substrates. The DNAzymes and ribozymes are introduced into the amplicons by using primers with 5′ tags that are inactive, anti-sense sequences of catalytic nucleic acids. When these sequences are copied during in vitro amplification the catalytically active sense sequences are co-amplified along with target sequence. The zymogene/DzyNA approach is very flexible since catalytic signal amplification can be linked to target amplification methods including PCR (polymerase chain reaction), strand displacement amplification (“SDA”), or rolling circle amplification (“RCA”), producing DNAzyme amplicons; and nucleic acid sequence-based amplification (“NASBA”), self-sustained sequence replication (“3SR”), or transcription-mediated amplification (“TMA”) amplification methods producing ribozyme amplicons. Further, since numerous catalytic nucleic acid molecules with a broad range of catalytic activities have been discovered or evolved, the zymogene approach can use a reporter substrate other than a nucleic acid where the readout of the assay is dependent on a chemical modification other than cleavage of a nucleic acid substrate. The zymogene/DzyNA (Todd et al., 2000) or NASBA/ribozyme (WO 00/58505) approach may be considered sensitive and useful, but there is potential for noise due to amplification of primer sequences.
NASBA has been used to produce RNA amplicons containing target nucleic acid and one section of the catalytic core of the hammerhead ribozyme (GAArA), introduced as antisense sequence tagged to a primer and then copied (WO 00/58505). The additional sequence required for catalytic activity (CUrGANrGrA) was introduced as sense sequence on a second molecule, which was labeled with a fluorophore and quencher, and which also served as the reporter substrate. Certain of the ribonucleotide bases (rN above) must remain as ribonucleotides, or catalytic ribozyme activity is lost. Two molecules consisting entirely of DNA were considered unable to form catalytically active heterodimer enzymes (WO 00/58505).
Catalytic nucleic acids have also been used for detection of single nucleotide polymorphisms (“SNPs”). The strict requirement for Watson Crick base pairing between the catalytic nucleic acid binding arms and the substrate has allowed the development of methods that allow discrimination of closely related short sequences. DNAzymes and ribozymes have been shown to discriminate between two sequences differing by as little as a single base (Cairns et al., 2000) (WO 99/50452).
DNAzymes have properties which provide advantages over ribozymes for certain in vitro applications. DNA is inherently more stable than RNA and hence is more robust with a longer shelf life. DNA can be stored for long periods at room temperature either in solution or in a lyophilized form. DNAzymes also are preferable over the majority of protein enzymes in certain applications because, for example, they are not irreversibly denatured by exposure to high temperatures during amplification.
MNAzymes can be used for nucleic acid quantification in conjunction with real-time polymerase chain reaction (PCR). Real time PCR, also abbreviated as Q-PCR, qPCR, QRT-PCR, or RT-qPCR, is a laboratory technique based on the PCR (polymerase chain reaction), to amplify and simultaneously quantify targeted DNA molecules. It enables both detection and quantification (as absolute copy numbers or relative amount of reference genes) of one or more specific sequences in a DNA sample. The procedure follows the general principle of polymerase chain reaction. The amplified DNA is detected as the reaction progresses in real time. Two other common methods for detection of products in real-time PCR are: (1) sequence-specific DNA probes consisting of oligonucleotides that are labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target, and (2) non-specific fluorescent dyes that intercalate with any double-stranded DNA. The commonly used reagent for method (1) is TaqMan probes and for method (2) is the SYBR Green I dye. Frequently, real-time PCR is combined with reverse transcription to quantify RNA (including messenger RNA and Non-coding RNA).
TaqMan probes are hydrolysis probes that are designed to increase the specificity of real-time PCR assays (Holland, P M; Abramson, R D; Watson, R; Gelfand, D H (1991). “Detection of specific polymerase chain reaction product by utilizing the 5′----3′ exonuclease activity of Thermus aquaticus DNA polymerase”. Proceedings of the National Academy of Sciences of the United States of America 88 (16): 7276-80. PMID 1871133; Gelfand, et al., U.S. Pat. No. 5,210,015; Mayrand; Paul E.: U.S. Pat. No. 7,413,708). TaqMan utilizes a dual-labeled probe (containing a fluorophore and matched fluorescence quencher) and fluorophore-based detection. During hybridization to the complementary target sequence, the 5′-3′ nuclease activity of Taq DNA polymerase releases the fluorophore from proximity to the quencher, generating fluorescence intensity proportionate to the amount of complementary target sequence in the reaction. As in other real-time PCR methods, the resulting fluorescence signal permits quantitative measurements of the accumulation of the product during the exponential stages of the PCR; however, the TaqMan probe significantly increases the specificity of the detection.
TaqMan probes consist of a fluorophore covalently attached to the 5′-end of the oligonucleotide probe and a quencher at the 3′-end. Several different fluorophores (e.g. 6-carboxyfluorescein, acronym: FAM, or tetrachlorofluorescin, acronym: TET) and quenchers (e.g. tetramethylrhodamine, acronym: TAMRA, or dihydrocyclopyrroloindole tripeptide minor groove binder, acronym: MGB) are available. The quencher molecule quenches the fluorescence emitted by the fluorophore when excited by the cycler's light source via FRET (Fluorescence Resonance Energy Transfer). As long as the fluorophore and the quencher are in proximity, quenching inhibits any fluorescence signals.
TaqMan probes are designed such that they anneal within a DNA region amplified by a specific set of primers. As the Taq DNA polymerase extends the primer and synthesizes the nascent strand, the 5′ to 3′ exonuclease activity of the polymerase degrades the probe that has annealed to the template. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR.
Another commonly used reagent for detection of products in real-time PCR is SYBR Green I (SG), an asymmetrical cyanine dye that is also used as a nucleic acid stain in molecular biology. SYBR Green I binds to double-stranded DNA. The resulting DNA-dye-complex absorbs blue light (λmax=488 nm) and emits green light (λmax=522 nm). SYBR Green I can be used for real-time PCR detection because there is a linear relation between the double-stranded DNA synthesized and the amount of green light emitted.
TaqMan requires producing double-labeled probes specific for each product, which can increase the cost of TaqMan-based real-time PCR system. However, unlike SYBR Green I, TaqMan can readily be utilized for multiplex PCR since a reaction can contain multiple TaqMan probes, each specific for a particular amplicon and each utilizing a distinguishable fluorophore.
As mentioned above, the MNAzyme system can also be utilized for product detection in real-time PCR. The inactive subunits can each contain a sensor arm complementary to a portion of the PCR product, together with a substrate-recognition arm complementary to a portion of a double-labeled probe (containing a fluorophore and matched quencher). Multiplex PCR can be achieved by utilizing multiple MNAzymes that differ in their sensor arm (conferring recognition of different PCR products) and also differ in their substrate recognition arms (conferring cleavage of different probes containing distinguishable fluorophores), such that accumulation of each PCR product leads to release of a particular fluorophore. However, MNAzymes have the potential advantage that double-labeled probes containing amplicon-specific sequences are not required; rather, because probe cleavage is mediated by the MNAzyme through its substrate recognition arm, “universal” probes can be utilized. By avoiding the need to synthesize double-labeled probes specific for each amplicon, MNAzyme has the potential to be more economical than TaqMan.
Additional background information concerning MNAzymes and other pertinent information may be found in the following documents, each of which is incorporated by reference in its entirety US 20100221711; US 20100136536; US 20100035229; US 20070231810; Nauwelaers D, Vijgen L, Atkinson C, Todd A, Geretti A M, Van Ranst M, Stuyver L. Development of a real-time multiplex RSV detection assay for difficult respiratory samples, using ultrasone waves and MNAzyme technology. J Clin Virol. 2009 November; 46(3):238-43. Epub 2009 Sep. 15; Gerasimova Y V, Kolpashchikov D M. Nucleic acid detection using MNAzymes. Chem. Biol. 2010 Feb. 26; 17(2):104-6; Teller C, Willner I. Functional nucleic acid nanostructures and DNA machines. Curr Opin Biotechnol. 2010 Aug. 18. [Epub ahead of print]; WO/2007/041774; and Mokany E, Bone S M, Young P E, Doan T B, Todd A V. MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches. J Am Chem. Soc. 2010 Jan. 27; 132(3):1051-9.